Apoptosis, or programmed cell death, is a naturally occurring process that has been strongly conserved during evolution to prevent uncontrolled cell proliferation. This form of cell suicide plays a crucial role in the development and maintenance of multicellular organisms by eliminating superfluous or unwanted cells. However, if this process goes awry, excessive apoptosis results in cell loss and degenerative disorders including neurological disorders such as Alzheimer's disease, Parkinson's disease, ALS, retinitis pigmentosa and blood cell disorders, while insufficient apoptosis contributes to the development of cancer, autoimmune disorders and viral infections (Thompson, Science, 1995, 267, 1456-1462).
Although several stimuli can induce apoptosis, little is known about the intermediate signaling events, including inhibition, that connect the apoptotic signal to a common cell death pathway conserved across many species. Recently, major advances have been made in understanding the signaling pathways mediated by the tumor necrosis factor receptor (TNFR) family which signals apoptosis. Two cell surface cytokine receptors of the TNFR family, TNFR-1 and CD95 (Fas/APO-1), act as death receptors and a number of binding proteins have been identified which mediate apoptosis through these receptors.
FADD (also known as Fas associated death domain and MORT1, for mediator of receptor induced toxicity) is a protein which interacts with the cytoplasmic domain of Fas/APO-l acting as a downstream effector in the process of apoptosis (Chinnaiyan et al., Cell, 1995, 81, 505-512). Overexpression of FADD has been shown to induce apoptosis through the activation of cell proteases. Support for this conclusion comes from studies which show that CrmA, a poxvirus gene product which targets interleukin-1 beta converting enzyme (ICE), a pro-apoptotic protease, can suppress FADD-induced apoptosis (Chinnaiyan et al., Cell, 1995, 81, 505-512). In addition, FADD has been shown to mediate TNF-dependent activation of acid sphingomyelinase (A-SMase) (Schwandner et al., J. Biol. Chem., 1998, 273, 5916-5922), lipopolysacharide-induced apoptosis (Choi et al., J. Biol. Chem., 1998, 273, 20185-20188), embryonic development (Yeh et al., Science, 1998, 279, 1954-1958), and T-cell activation and development (Walsh et al., Immunity, 1998, 8, 439-449; Zhang et al., Nature, 1998, 392, 296-300).
Gene therapy technology has also been exploited in the study of FADD-mediated apoptosis. Studies designed to determine whether tumors resistant to Fas/APO-1 cytotoxicity could be rendered susceptible to apoptosis by the overexpression of FADD demonstrated that retroviral transfection of the FADD gene into malignant glioma cells induced apoptosis in 85% of the cells (Kondo et al., Hum. Gene Ther., 1998, 9, 1599-1608).
To date, strategies aimed at inhibiting FADD function have involved the use of dominant negative forms of the protein, and gene knock-outs in mice.
A dominant-negative form of FADD lacking the N-terminal 80 amino acids was shown to block CD95 and TNFR-1-induced apoptosis without affecting the TNFR-1 activation of NF-kB (Chinnaiyan et al., J. Biol. Chem., 1996, 271, 4961-4965). Furthermore, using the dominant-negative FADD proteins described by Chinnaiyan (Chinnaiyan et al., Cell, 1995, 81, 505-512; Chinnaiyan et al., J. Biol. Chem., 1996, 271, 4961-4965), Herr et al. demonstrated that the second messenger, ceramide, links cellular stress responses induced by .gamma.-radiation or anticancer drugs to the CD95 pathway of apoptosis (Herr et al., Embo J., 1997, 16, 6200-6208).
Finally, mice expressing another dominant-negative mutant, lacking amino acids 80-208 comprising the death domain, showed enhanced deletion of autoreactive thymocytes and inhibition of mature T lymphocyte proliferation (Newton et al., Embo J., 1998, 17, 706-718).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of FADD. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting FADD function. Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of FADD expression.